Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.

We present an efficient approach for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to rapidly create a library of complex functional ionizable lipopolymers. We synthesized a diverse library of 155 polymers, formulated them into polyplexes to establish structure-activity relationships crucial for endosomal escape and efficient transfection. After discovering a lead structure, lipopolymer-lipid hybrid nanoparticles are introduced to preferentially deliver to and elicit effective mRNA transfection in lung endothelium and immune cells, including T cells with low in vivo toxicity. The lipopolymer-lipid hybrid nanoparticles showed 300-fold improvement in systemic mRNA delivery to the lung compared to in vivo -JetPEI ® . Lipopolymer-lipid hybrid nanoparticles demonstrated efficient delivery of mRNA-based therapeutics for treatment of two different disease models. Lewis Lung cancer progression was significantly delayed after treatment with loaded IL-12 mRNA in U155@lipids after repeated i.v. administration. Systemic delivery of human CFTR (hCFTR) mRNA resulted in production of functional form of CFTR protein in the lungs. The functionality of hCFTR protein was confirmed by restoration of CFTR- mediated chloride secretion in conductive airway epithelia in CFTR knockout mice after nasal instillation of hCFTR mRNA loaded U155@lipids. We further showed that, U155@lipids nanoparticles can deliver complex CRISPR-Cas9 based RNA cargo to the lung, achieving 5.6 ± 2.4 % gene editing in lung tissue. Moreover, we demonstrated successful PD-1 gene knockout of T cells in vivo . Our results highlight a versatile delivery platform for systemic delivering of mRNA of various sizes for gene therapy for a variety of therapeutics.

Effect of Cysteine Residue Substitution in the GCSAG Motif of the PMGL2 Esterase Active Site on the Enzyme Properties.

The gene coding for PMGL2 esterase, which belongs to the family of mammalian hormone-sensitive lipases (HSLs), was discovered by screening a metagenomic DNA library from a permafrost soil. The active site of PMGL2 contains conserved GXSXG motif which includes Cys173 residue next to the catalytic Ser174. In order to clarify the functional role of the cysteine residue in the GCSAG motif, we constructed a number of PMGL2 mutants with Cys173 substitutions and studied their properties. The specific activity of the C173D mutant exceeded the specific activity of the wild-type enzyme (wtPMGL2) by 60%, while the C173T/C202S mutant displayed reduced catalytic activity. The activity of the C173D mutant with p-nitrophenyl octanoate was 15% higher, while the activity of the C173T/C202S mutant was 17% lower compared to wtPMGL2. The C173D mutant was also characterized by a high activity at low temperatures (20-35°C) and significant loss of thermal stability. The kcat value for this protein was 56% higher than for the wild-type enzyme. The catalytic constants of the C173S mutant were close to those of wtPMGL2; this enzyme also demonstrated the highest thermal stability among the studied mutants. The obtained results demonstrate that substitutions of amino acid residues adjacent to the catalytic serine residue in the GXSXG motif can have a significant effect on the properties of PMGL2 esterase.

Preparation and Testing of Cells Expressing Fluorescent Proteins for Intravital Imaging of Tumor Microenvironment.

Intravital microscopy is widely used for in vivo studies of the mechanisms of carcinogenesis and response to antitumor therapy. For visualization of tumor cells in vivo, cell lines expressing fluorescent proteins are needed. Expression of exogenous proteins can affect cell growth rate and their tumorigenic potential. Therefore, comprehensive analysis of the morphofunctional properties of transduced cells is required for creating appropriate models of tumor microenvironment. In the present study, six lines of mouse tumor cells expressing green and red fluorescent proteins were derived. Analysis of cells morphology, growth kinetics, and response to chemotherapy in vitro revealed no significant differences between wild-type and transduced cell lines. Introduction of fluorescent proteins into the genome of 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells did not affect tumor growth rate after subcutaneous implantation to mice, while both CT26-GFP and CT26-RFP cells (murine colon cancer) were rejected starting from day 8 after implantation. Elucidation of the mechanisms underlying CT26-GFP/RFP rejection is required to modify transduction technique for creating the models of tumor microenvironment accessible for in vivo visualization. Transduced 4T1 and B16-F10 cell lines can be used for intravital microscopic imaging of tumor cells, neoplastic vasculature, and leukocyte subpopulations.

ENALAPRIL EFFECT ON THE STATE OF NITROGEN OXIDE SYSTEM AND PROOXIDANT-ANTIOXIDANT BALANCE IN BRAIN UNDER CONDITIONS OF BLOCKADE OF CENTRAL CHOLINERGIC SYSTEM.

One of the most important medical-social issues today is the search for new pathogenic directions in pharmacological prevention and treatment of neurodegenerative diseases of the central nervous system. Objective: to investigate enalapril effect on the indices of the nitrogen oxide system and prooxidant-antioxidant balance in the cerebral cortex and hippocampus under conditions of experimental cerebral neurodegeneration. The state of the nitrogen oxide system and prooxidant-antioxidant balance is investigated in the experiment on male rats with scopolamine-induced neurodegeneration (1 mg/kg, 27 days) after enalapril administration in the dose of 1 mg/kg (14 days). Analysis of the obtained data enabled to describe a possible mechanism of enalapril neuroprotective action. It is manifested in decrease of NO2 concentration (1,4 times in both examined structures) and NOS activity (1,6 times in the hippocampus), which is indicative of an increased synthesis of NO as endothelial vasodilator. Correspondingly, a modulating effect on NO system and prooxidant-antioxidant balance (the content of products reacting with 2-thiobarbituric acid 1,1 times decreased in the cerebral cortex, and 1,3 times in the hippocampus compared with untreated rats; catalase activity in the cerebral cortex did not differ reliably from that of the index in the control group, and 1,4 times increased compared with untreated rats) can be considered as constituent mechanisms of enalapril neuroprotective effect under conditions of experimental scopolamine-induced neurodegeneration.

Correction: New ferrocene-based 2-thio-imidazol-4-ones and their copper complexes. Synthesis and cytotoxicity.

Correction for 'New ferrocene-based 2-thio-imidazol-4-ones and their copper complexes. Synthesis and cytotoxicity' by D. A. Guk et al., Dalton Trans., 2018, DOI: 10.1039/c8dt03164a.

New ferrocene-based 2-thio-imidazol-4-ones and their copper complexes. Synthesis and cytotoxicity.

Synthesis, characterization (HRMS, NMR, EPR, XANES, UV-Vis spectroscopy, and electrochemistry), DNA and BSA binding and in vitro biological screening of two new ferrocene-incorporated thiohydantoin derivatives (5 and 6) and their copper coordination compounds are reported. The ferrocene-based thiohydantoin derivatives were prepared by copper-catalyzed azide alkyne cycloaddition reactions between alkynyl ferrocenes and 5-(Z)-3-(2-azidoethyl)-2-(methylthio)-5-(pyridin-2-ylmethylene)-1H-imidazol-4H-one. Alkynyl ferrocenes necessary for these syntheses were prepared by new procedures. Intermolecular redox reactions between the ferrocene fragment and copper(+2) coordinated ions were studied by different methods to determine the mechanism and kinetic constants of redox processes. Ferrocene-containing imidazolones (5 and 6) and their copper complexes were also tested for their in vitro cytotoxic activity against MCF-7 and A-549 carcinoma cells, and also against the noncancerous cell line Hek-293. The results showed modest cytotoxicity against the subjected cancer cell line compared with cisplatin. The ability of the obtained compounds to cause DNA degradation and cell apoptosis was investigated, and the distribution of cytosol/pellets was studied by AAS.

High-sensitive absorption measurement in transparent isotropic dielectrics with time-resolved photothermal common-path interferometry.

We propose a time-resolved photothermal common-path interferometry scheme at fast heating in the absence of heat diffusion and employ it to measure absorption in Suprasil 311 silica glass (2.8·10-6 cm-1) at a wavelength of 1071 nm and continual absorption in laboratory air (2.9·10-8 cm-1) for a signal-to-noise ratio of 100/1. The absorption was measured at a thermally induced phase incursion of less than 0.1 rad in a heating beam, which guaranteed correct calibration. To calibrate this scheme, we developed a theory of diffraction on deformations taking into account the stresses arising in an inhomogeneous temperature field. This allowed us to use a standard glass K8 for calibration. The low level of noise and time resolution of pulsed signals allowed the distinguishing of the contributions of Kerr and striction nonlinearities to absorption measurements in Suprasil 311 silica glass and enabled the observance of the time evolution of strictional deformations. Additionally, an anomalous temporal development of the absorption of broadband laser radiation in atmospheric air at 2.9·10-8 cm-1 has been revealed.

Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli.

Cell surface display is a popular approach for the construction of whole-cell biocatalysts, live vaccines, and screening of combinatorial libraries. To develop a novel surface display system for the popular scaffold protein 10th human fibronectin type III domain (10Fn3) in Escherichia coli cells, we have used an α-helical linker and a C-terminal translocator domain from previously characterized autotransporter from Psychrobacter cryohalolentis K5T. The level of 10Fn3 passenger exposure at the cell surface provided by the hybrid autotransporter Fn877 and its C-terminal variants was low. To improve it, the fusion proteins containing 10Fn3 and the native autotransporter passenger Est877 or the cold-active esterase EstPc in different orientations were constructed and expressed as passenger domains. Using the whole-cell ELISA and activity assays, we have demonstrated that N-terminal position of EstPc in the passenger significantly improves the efficiency of the surface display of 10Fn3 in E. coli cells.

[Exposure to toxic dose of adrenaline on the functional state of the liver].

The purpose: The blood biochemical parameters characterizing the functional state of the liver, and the morphological profile of the body after a single exposure to a toxic dose of adrenaline were studied. Methods: Studies were conducted on 60 adult rats (female) weighing 0.15-0.2 kg, were divided into groups: intact animals; experience - animals, injected with epinephrine hydrochloride intraperitoneally in a dose of 0.5 mg/kg. All kinds of Biological material (blood, liver) were collected out through one and ten days after the start of the experiment. The degree of influence of high doses of epinephrine were evaluated in terms of lipid peroxidation (LPO) and protein (PSP) in liver homogenates, the concentration of average weight molecules (MSM), the activity of ALT, AST, alkaline phosphatase, LDH, total protein concentration, glucose and lactate in the blood plasma, as well as the determination of the prothrombin time (PTT) with the counting on the basis thereof of international normalized ration (INR). Histology of the liver was studied by light microscopy. Results: It was found that throughout the experiment, there was an increased in the concentration of lipid peroxidation products and protein in liver homogenates, there was an increase in the concentration of MSM 1.7. Twenty-four hours after the administration of a toxic dose of adrenaline observed hyperenzymemia that manifested an increase in the activity of ALT and AST, was an increase in LDH. After 10-day five after the start of the experiment established the presence hyperenzymemia activity decreased ALT and AST, LDH activity remained elevated, total protein level was higher than in the group of animal in which investigations were conducted one day after the start of the experiment, PTV also continued to decline. In histological sections of the development of a pathological condition characterized by circulatory disturbance - plasmatization, both in central and in small vessels. From the hepatocytes both in the center and the periphery had changes granular dystrophy type, to some extent vacuolar.

Sub-terahertz microsecond optically controlled switch with GaAs active element beyond the photoelectric threshold.

We study an unusual working regime of a recently developed sub-terahertz microwave cavity-based switch. The resonator cavity includes a semiconductor plate which is illuminated by laser emission beyond the photoelectric threshold. Despite a significant change to the conventional process of photoelectric effect we have found that the switch works. Typical switching performance rate is about 1 µs for the regime. A process of carrier density relaxation beyond the photoelectric threshold is discussed. An idea of diagnostic method for the semiconductor's quality is proposed.

Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5(T).

We have cloned the gene coding for AT877-a new predicted member of the autotransporter protein family with an esterase passenger domain from permafrost bacterium Psychrobacter cryohalolentis K5(T). Expression of AT877 gene in Escherichia coli resulted in accumulation of the recombinant autotransporter in the outer membrane fraction and at the surface of the induced cells. AT877 displayed maximum hydrolytic activity toward medium-chain p-nitrophenyl esters (C8-C10) at 50 °C and was resistant to the presence of several metal ions, organic solvents and detergents. Previously, we have described a cold-active esterase EstPc from the same bacterium which possesses high activity at low temperatures and relatively high thermal stability. To construct a cell surface display system for EstPc, the hybrid autotransporter gene coding for EstPc with the α-helical linker and the translocator domain from AT877 was constructed and expressed in E. coli. According to the results of the cell fractionation studies and esterase activity measurements, the EstPc passenger was successfully displayed at the surface of the induced cells. It demonstrated a temperature optimum at 15-25 °C and a substrate preference toward p-nitrophenyl butyrate (C4). Obtained results provide a new example of the biotechnologically relevant enzyme from the permafrost microbial community with potential applications for the conversion of short- and medium-chain ester substrates and a basis for the construction of a new cell surface display platform.

Distinct biological activity of lipopolysaccharides with different lipid A acylation status from mutant strains of Yersinia pestis and some members of genus Psychrobacter.

Correlation between the chemical structure of lipid A from various Gram-negative bacteria and biological activity of their lipopolysaccharide (LPS) as an agonist of the innate immune receptor Toll-like receptor 4 was investigated. Purified LPS species were quantitatively evaluated by their ability to activate the production of tumor necrosis factor (TNF) by murine bone marrow-derived macrophages in vitro. Wild-type LPS from plague-causing bacteria Yersinia pestis was compared to LPS from mutant strains with defects in acyltransferase genes (lpxM, lpxP) responsible for the attachment of secondary fatty acid residues (12:0 and 16:1) to lipid A. Lipid A of Y. pestis double ΔlpxM/ΔlpxP mutant was found to have the chemical structure that was predicted based on the known functions of the respective acyltransferases. The structures of lipid A from two members of the ancient psychrotrophic bacteria of the genus Psychrobacter were established for the first time, and biological activity of LPS from these bacteria containing lipid A fatty acids with shorter acyl chains (C10-C12) than those in lipid A from LPS of Y. pestis or E. coli (C12-C16) was determined. The data revealed a correlation between the ability of LPS to activate TNF production by bone marrow-derived macrophages with the number and the length of acyl chains within lipid A.

Characterization of a cold-active lipase from Psychrobacter cryohalolentis K5(T) and its deletion mutants.

A gene coding for cold-active lipase from the psychrotrophic Gram-negative bacterium Psychrobacter cryohalolentis K5(T) isolated from a Siberian cryopeg has been cloned and expressed in Escherichia coli. The recombinant protein Lip1Pc with a 6× histidine tag at its C-terminus was purified by nickel affinity chromatography. With p-nitrophenyl dodecanoate (C12) as a substrate, the purified recombinant protein displayed maximum lipolytic activity at 25°C and pH 8.0. Increasing the temperature above 40°C and addition of various metal ions and organic solvents inhibited the enzymatic activity of Lip1Pc. Most nonionic detergents, such as Triton X-100 and Tween 20, slightly increased the lipase activity, while SDS completely inhibited it. To investigate the functional significance of the Lip1Pc N-terminal domain, we constructed five deletion mutants of this protein. The ND1 and ND2 mutants displayed specific activity reduced by 30-35%, while other truncated proteins were completely inactive. Both mutants demonstrated increased activity towards p-nitrophenyl decanoate (C10) and impaired utilization of C16 substrate. Although optimum reaction temperature of ND2 lowered to 20°C, it displayed enhanced stability by 44% after incubation at 40°C. The results prove that the N-terminal domain of Lip1Pc has a fundamental impact on the activity and stability of the protein.

[Molecular model of binding of estradiol and 8-isoestradiol with estrogen alpha-receptor]. / Izuchenie sviazyvaniia éstradiola i 8-isoéstradiola s al'fa-retseptorom éstrogenov metodom moleculiarnogo modelirovaniia.

The complexes of the estrogen alpha-receptor with estradiol and 8-isoestradiol were comparatively analyzed. The computations of ligand-receptor complexes, carried out using the FLEXX program, allowed us to propose a model for the binding of the analogues of 8-isoestradiol. It was found that rings C and D of estradiol and 8-isoestradiol are similarly arranged in the ligand-binding pocket and coincide upon the superposition of the corresponding ligand-receptor complexes, whereas rings A and B do not coincide. The oxygen functions in position 17 of the estradiol analogues of both series coincide upon superposition, whereas the phenol 3-hydroxyl groups are 0.05 A apart. A comparison of the predicted biological properties of modified estradiol analogues of the natural and 8-isoseries with the available experimental data revealed their similarity. Synthetic 2-acetyl analogues of 8-isoestrogens were found to have no uterotropic activity, which is also consistent with the proposed model.